Efficient non-viral T cell engineering for TCR gene therapy by Sleeping Beauty minicircles

Sleeping Beauty (SB) Transposon-basierte Vektoren werden als Alternative zu viralen Vektoren für T-Zell-Gentherapie erforscht und ermöglichen eine schnelle und kostengünstige Genmanipulation von T-Zellen. Die Verwendung von Transposon-Vektoren erfordert jedoch die DNA-Elektroporation von T-Zellen, die sich schädlich auf T-Zellen auswirkt. DNA-elektroporierte T-Zellen weisen eine verringerte Lebensfähigkeit und eine verzögerte Aktivierung nach Stimulation des T-Zell-Rezeptors (TCR) auf. Um die Nachteile der Transposon-basierten T-Zell-Genmanipulation zu überwinden, haben wir neuartige SB-Vektoren entwickelt. Durch die Kombination von SB Transposon-basierten Minicircle-Vektoren mit SB100X Transposase-mRNA konnten T-Zellen effizient genmodifiziert werden. Unser Ansatz reduzierte die T-Zell-Mortalität und steigerte gleichzeitig die Transfektionseffizienz. Mit diesen neuartigen Vektoren wurde die stabile Expression verschiedener TCRs und CARs in über 50% der eingesetzten T-Zellen erreicht. Gentechnisch manipulierte T-Zellen konnten Antigen-spezifisch stimuliert werden und zeigten effiziente Zytokin-Sekretion und Tumorzell-Lyse. Weiterhin haben wir miRNAs entwickelt, die die Expression der endogenen TCR-Ketten unterdrücken. Der Einbau dieser miRNAs in die TCR-Expressionskassette erhöhte die Oberflächenexpression des therapeutischen TCRs, verringerte die Fehlpaarung mit endogenen TCR-Ketten und erhöhte die T-Zell-Funktionalität. Ein direkter Vergleich von SB- und Virus-modifizierten T-Zellen zeigte sowohl in vitro als auch in vivo eine vergleichbare Wirksamkeit der modifizierten T-Zellen hinsichtlich Zytokin-Sekretion, Tumorzell-Lyse und Tumorkontrolle. In dieser Arbeit konnte gezeigt werden, dass SB Minicircle-Vektoren die Herstellung von genetisch modifizierten T-Zellen ermöglichen und diese Tumor-spezifische Wirksamkeit aufweisen. Dieser Ansatz könnte die Herstellung therapeutischer T-Zellen für die personalisierte T-Zell-Gentherapie vereinfachen und beschleunigen.Sleeping Beauty (SB) transposon-based vectors have entered clinical trials as an alternative to viral vectors for T cell gene therapy, offering time- and cost-efficient engineering of therapeutic T cells. However, transposon vectors require DNA electroporation into T cells, which we found to cause adverse effects. T cell viability was decreased, and DNA-transfected T cells showed delayed activation upon T cell receptor (TCR) stimulation regarding blast formation and proliferation. To overcome the limitations of transposon-based T cell engineering, we investigated the effect of DNA electroporation on T cells and developed novel SB vectors. T cells could efficiently be engineered with Sleeping Beauty vectors by combining SB transposon minicircles and SB100X transposase mRNA. Our approach reduced T cell mortality and substantially enhanced transfection efficiency. We achieved stable expression of several TCRs and CARs in more than 50% of the transfected T cells compared to 15% when conventional plasmids were used. T cells engineered to express a tumor-specific TCR mediated effective tumor cell lysis and cytokine secretion upon antigen-specific stimulation. Furthermore, we developed miRNAs to silence the expression of the endogenous TCR chains. Incorporation of these miRNAs into the TCR expression cassette increased surface expression of the therapeutic TCR, diminished mispairing with endogenous TCR chains, and enhanced T cell functionality. Importantly, a direct comparison of SB minicircle- and RV-engineered T cells in vitro as well as in vivo demonstrated equal T cell efficacy with regards to cytokine release, tumor cell lysis and tumor control. We demonstrated that SB minicircles enable the generation of gene-modified T cells with tumor-specific reactivity. Our approach facilitates the manufacturing of therapeutic T cells with superior biosafety and accelerates the generation of patient-specific T cell products for personalized T cell gene therapy

Phase-space structure of resonance eigenfunctions for chaotic systems with escape

Physical systems are usually not closed and insight about their internal structure is experimentally derived by scattering. This is efficiently described by resonance eigenfunctions of non-Hermitian quantum systems with a corresponding classical dynamics that allows for the escape of particles. For the phase-space distribution of resonance eigenfunctions in chaotic systems with partial and full escape we obtain a universal description of their semiclassical limit in terms of classical conditional invariant measures with the same decay rate. For partial escape, we introduce a family of conditionally invariant measures with arbitrary decay rates based on the hyperbolic dynamics and the natural measures of forward and backward dynamics. These measures explain the multifractal phase-space structure of resonance eigenfunctions and their dependence on the decay rate. Additionally, for the nontrivial limit of full escape we motivate the hypothesis that resonance eigenfunctions are described by conditionally invariant measures that are uniformly distributed on sets with the same temporal distance to the quantum resolved chaotic saddle. Overall we confirm quantum-to-classical correspondence for the phase-space densities, for their fractal dimensions, and by evaluating their Jensen–Shannon distance in a generic chaotic map with partial and full escape, respectively.Typische physikalische Systeme sind nicht geschlossen, sodass ihre innere Struktur mit Hilfe von Streuexperimenten untersucht werden kann. Diese werden mit Hilfe einer nicht-Hermiteschen Quantendynamik und deren Resonanzeigenzuständen beschrieben. Die dabei zugrunde liegende klassische Dynamik berücksichtigt den Verlust von Teilchen. Für die semiklassische Phasenraumverteilung solcher Resonanzeigenzustände in chaotischen Systemen mit partieller und voller Öffnung entwickeln wir eine universelle Beschreibung mittels bedingt invarianter Maße gleicher Zerfallsrate. Für partiellen Zerfall stellen wir eine Familie bedingt invarianter Maße mit beliebiger Zerfallsrate vor, welche auf der hyperbolischen Dynamik und den natürlichen Maßen der vorwärts gerichteten und der invertierten Dynamik aufbauen. Diese Maße erklären die multifraktale Phasenraumstruktur der Resonanzzustände und deren Abhängigkeit von der Zerfallsrate. Darüber hinaus motivieren wir für den nicht trivialen Grenzfall voll geöffneter Systeme die Hypothese, dass Resonanzeigenzustände durch ein bedingt invariantes Maß beschrieben werden, welches gleichverteilt auf solchen Mengen ist, die den gleichen zeitlichen Abstand zum quantenunscharfen chaotischen Sattel haben. Insgesamt bestätigen wir die quantenklassische Korrespondenz für die Phasenraumdichten, deren fraktale Dimensionen und durch Auswertung ihres Jensen–Shannon Abstandes in einer generischen chaotischen Abbildung sowohl für partielle als auch für volle Öffnung

BIOMECHANICAL SKATING TECHNIQUE ANALYSIS IN BIATHLON

The paper describes examples from a biomechanical research project which is carried out in co-operation between the German Skiing Association/national team in biathlon and the University of Leipzig. The aims of the study were a) to develop a practical procedure to verify dynamic and kinematic skating technique features under typical conditions for the sport, b) to determine valid skating technique characteristics and c) to develop an feedback procedure which can be used in training sessions. The findings have been applied in training during the preparatory phase for the competition season 1999/2000. Biomechanically the study focussed on the determination of an effective temporal coordination of system propulsion impulses being generated by arms/poles and legs/skis

BIOMECHANICAL ANALYSIS OF THE DYNAMICS OF SKATING (SKATING 1-2)

Biomechanical data (curves and values) on skating technique are necessary for effective technique training. 1995-1997 kinematographic 3Danalyses of international top class athletes were made during uphill skating. A kinematic description of skating technique, as well as of technical reserves of top German athletes was obtained (Herrmann/Clauß, 1997). The specific dynamic reasons for the kinematic appearance were the subject of continued analyses. It was also aimed to collect information about measuring technical requirements of specific sensors and their attachment to the specific ski and pole. Corresponding results published so far are insufficient and affected by problems

Anisotropic elastic properties of common ash (Fraxinus excelsior L.)

Hardwoods in principle show a similar orthotropic behavior as softwoods; however, the ratios of the mechanical parameters between the three anatomical directions and their magnitudes are different and depend strongly on the individual microstructure of the species. The aim of the current study was to characterize the 3-D elastic behavior of common ash (Fraxinus excelsior L.) by tensile, compression, and shear tests in the three anatomical directions and stepwise in between, by means of a universal testing machine in combination with a digital image correlation technique. Young's moduli, shear moduli, and Poisson's ratios have been determined for the different load directions. From studies on the radial-tangential plane of other wood species, it is known that the elastic moduli in the principal directions and the off-axis elastic moduli vary in a nonlinear correlation, depending on density gradients between earlywood and latewood. This angular dependency has been experimentally and theoretically proven for ash. Furthermore, the dependency of mechanical parameters on the fiber-load angle has been experimentally determined. The measurements for principal and off-axis load directions provide a sound basis for modeling of hardwood structure

Stability analysis of multiplayer games on adaptive simplicial complexes

We analyze the influence of multiplayer interactions and network adaptation on the stability of equilibrium points in evolutionary games. We consider the Snowdrift game with both two-player and three-player interactions on simplicial complexes. The state of the system and the topology of the interactions are both adaptive through best response strategies of nodes and rewiring strategies of edges, respectively. We derive a closed set of low-dimensional differential equations using pairwise moment closure, which yields an approximation of the lower moments of the system. We numerically confirm the validity of these moment equations. Moreover, we demonstrate that the stability of the fixed points remains unchanged for the considered adaption process. This stability result indicates that rational best response strategies in games are very difficult to destabilize, even if higher-order interactions are taken into account

Procurement in 21st century in the chemical, pharmaceutical and healthcare industry

Most companies in the Chemical, Pharmaceutical and Healthcare industry have a procurement performance below average, which in turn means a disadvantage compared to other industries. These companies are not able to realize their existing cost reduction potentials in procurement. Additional short term potentials will not be fully exploited, for example these due to the financial crisis. A large fraction of companies from this industry has neither a relevant procurement strategy nor a procurement controlling. It is easy to see, that companies neglecting these basic elements have no chance to achieve a high procurement performance and optimize potentials. One specific problem is that the industry lacks of sufficient qualified procurement personnel. At the same time important positions in procurement departments, especially in strategic procurement, were vacant. In general procurement in the Chemical, Pharmaceutical and Healthcare industry has a strong operational focus. Highest prioritized topics are analysis of prices and offers, calculation and planning of demands and management of supply shortages. In comparison to other industries each of these topics has relevance above-average. The major topics in strategic procurement are evaluation of suppliers, tendering and qualification of suppliers as well as searching for new suppliers. Especially tendering seems to be more relevant for companies in the Chemical, Pharmaceutical and Healthcare industry than for companies in other industries. But still companies in the Chemical, Pharmaceutical and Healthcare industry do not fully benefit from maximum cost reduction effect from tendering.<br

Thermal stability of glued wood joints measured by shear tests

The thermal stability of glued wood joints is an important criterion to determine the suitability of adhesives in the field of engineered wood. During their product life, glued wood joints can be exposed to high temperatures in various ways (direct exposure to the sun, fire, etc.). Thereby the cohesiveness of the adhesive must not degrade. This raises the question of how the strength of bonding changes under thermal load. The current investigation covers the influence of temperature (T=20 to 220°C) on the shear strength of glued wood joints. Different adhesive systems were investigated. With increasing temperature, the shear strength of solid wood and also of glued wood joints decreased. There were big differences in thermal stability and failure behaviour between the adhesive systems as well as within the polyurethane group. The thermal stability of one-component polyurethane systems can be greatly varied by modifying their chemical structure. Well adapted one-component polyurethane adhesives reach a strength similar to that of phenol resorcinol resi